Medical serum



June 25,1929. FEJEs' EIAL 1.718.282

MEDI CAL SERUM Filed Feb; 16 1927 Patented June 25,1929;

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. L-Unwm' rams AND BRUNO nh -r'rcnnn, or 3mm, omen, assronoas 7530 THE rum anmno-osuosn axrmnensnmsonm (em-minim eEsELLscmr OI BERLIN, GERMANY.

. MEDICAL smum.

application flled lcbruary 1a,'1m. Serial m.,1 a,m

This invention refers broadly to improveu The most undesirable component isthe sec-' 55 0nd globuline' fraction,-the'so-called euglob ments in medical sera, and the object of the invention is a serum of'higher purity than obtainable heretofore andthe administration of which is free from the noxious actions frequentlyaccompanying the administration of the products heretofore in use. The invention is based uponthe utilization ofthe immune serum obtained from correspondingly treated animals and purified by means of an electro-osmotic procedure, such as is for'instance described .in the copending a plication, 14,77 6 for U. S. patent, filed by ilhelm Ruppel. By means of this electro-osmotic method of purification a product is obtained.

which is of a higher degree of purity than obtainable heretofore. In accordance with the previous art it was-the object to remove from the so-called native or crude sera 'iurnished by the immunized animal system, all those substances which arenot conducive to the medical or curative effect pro er. These components generally designate as.

- ballast-substances contain fractions of proobjectionable increase of the total contents.

tein, which, while being free from injurlous properties themselves, fail to contain immune substances and by their presence produce an of protein of the serum. Of these protein fractions the albumin-bodies should be par ticularly noted, and of a similar nature, as regards the purposes ofv this invention, are the: decomposition products contained in every serum, the amino-acids, the invariably present lipoids, coloring matter, purin bases and the like.

' On the other hand however, and in com tradistinction'th'erefrom the globulines con tained in the serum cannot be regarded the l equivalent of the ballast substances referred to. In' acc'o'rdance with the present state of theart it has been generally admitted that the most; prominent constituents of these bodies, that'is to 'say the pseudo-globulines, represent. the carriers of the actual curative medicinal substances of the specific sera; In the case of the diplitheria-curative-serum which istheserum most easily accessible to investigation it can be readily ascertained that more than 90% of the ant-itoxine-units contained .in the finished serum are incorporated in this fraction of the serum and the same holds true of 'all the other antitoxic and antibacterial immune sera.

uline. The euglobul-ines, while containing all those antltoxme or immune'bodie's which,

aside from those incorporated with the pseue A doglobuline, are to be found in the serum,

are the specific carriers of all those bodies which are the cause of the objectionable, lin

desirable, andev'en injurious properties of cause anaphylaxia upon repeated injection and the so-called serum-disease. ,In the.

the curative seraf. In the euglobuline-frac- .t on those substances areme't with which euglobulines the antigen-residues will more over-remain, that is. to say, parts of the po sons employed for the immunization of the blood-furnlshing horses against the ;ac.-

tion of which-the animal system is unablemore, the presence of the euglobulinesthe c'auseof the weakening of the curative 'sera. B the action of the euglobulinespres'entam to ytic processes are produced in the serum 7 in the course of time which are always ac]- compamed by the decomposition of the valu able serum-components, .and of the pseudoglobulines in particular above referred to,

so as to result in munizingbodies.

All purification processes eaturesthe salting out of subsequent dialysis are object1onable for the reason that by such methodstajseparation of the globulinefraction into pseudo-globulines and euglobulines cannot *be obtained,- so that the elimination 0f-theraction1astmentioned is not possible therebyg Only by the process of'electro-osmosis, and particularly by the methodhereinbefore referred to, and set forth in eopending applicathe destrubtion of the im- Meat an;

revious art and comprising in their essential eglobulines with tion 14,776 "of WilhehnRuppel it has become possible to arrive at a substantially quantitative splitting up of the g'lobuline mixtures into the components pscudoglobulines and englohulines, so that the latter'rnay be entirely removed thereby. v

On the accompanying drawings we have shown byway of example and merely diaratus in which the process of grammatically in vertical section an appapurification of the product, 'formin the sub ect of our in vention, may be per orin'ed.

In the drawing A indicates the anode chamber, K the cathode chamber, p and n are respectively the positive diaphragm and the negative diaphragm which are spatially disosed, so as toconstitute a central chamber M etween them. The anodic diaphragm preferably consists of vegetable material, canvas or the like,zwhile the cathodic" diaphragm comprises an animal membrane, gelatine or the equivalent thereof. The raw material,

blood serum, exudates or transudates is introduced into the central chamber and is submitted to the action of the direct current. By this means all the electrolytes are withdrawn,- and at the isoelectrie point (corresponding to a hydrogen-ion value of 6.4-P the insoluble v euglobulin is separated out which carries down all the antigens and the disease germs originally contained in the crude, native liq uids. The deposit, the. euglobuline, is then separated by filtration, centrifugation or otherwise from the liquid which contains the pseudoglobulines and the albumines. By the well known addition of salt these two bodies are separated. The precipitate obtained and consisting of pseudoglobulines is dissolved in water, and is again subjected to the action of j The purification may also be carried out by eliminating the alumbine and the bulk of the electrolytes and of the ballast substances, blood salts, carbon-hydrates, nitrogenous organic substances, amino-acids and peptones by salting out and filtration. The precipitate which contains mainly globulines and precipitants, is preferably dissolved I in a small Total proteids 8. 52%

- Albumine 1. 93%

Globuline' a. 6. 59% =4. 60% pseudoglobuline+1.9 8% euglobuline Pseudoglobuline-solution 11.02% p v I 1 Yield pf antit0xine-units 90% amount of-water, is treated with bariumacetate for the removal of sulphuric acid and sulphates, and is filtered off from the bariumsulphate. The clear. lobuline solution "obtained is then treated in therthree-cell apparatus in the manner described by electroosmosis, and the -albuminous :"a'nd .proteid bodies which have bepome fiocculate'd out Concentration factor- 1. 38%

(0.85% NaCl) a more or less concentratedtable and animal. membranes, causingthe albumines to be discharged as the anion while the globuline remains in the cathodic chamber and is separated into pseudoglobuline and euglobuline. I

EwampZes.-Ten liters diphtheria-serum are diluted with about'half as'much water and are treated Wit-habout the same quantity of saturated solution of ammonium-sulphate. After allowing to stand 6 to 10 hours, thesalted out globuline is sufliciently agglo'merated to allow of beingfiltered by hardened filters moved rom the filters and is dissolved in a small amount of water. 'Ehe amount'of ammonium sulphate is determined by titration of a small amount of the solution, after 00- 1 agulation by boiling, with barium-acetate,- then barium-acetate is added, the insoluble centrifugation. The clear solution containsthe globulines and small residues of bloodsalts and ammonium-acetate and *albuinine decomposition products. It'is treated in the apparatus described at a-current intensity of 10-15. ampere and at 2030 volts. After a few hours the current strength drops down 1 to 0.2 0.5 amps, while the voltage rises to 200- to 220 volts. At a hydrogen concentration of about 6.4 P a maximum of a flocculent precipitate, is formed, consisting of euglobuline' which is separated off by centrifugation, while the pseudoglobuline which is free from electrolytes may be deca'ntedofi.

' This pseudoglobuline which is free from' electrolytes constitutes in a faintly alkaline solution (7 .4 P withisotonic salt-contents product as compared with the original globuline solution as regards the-antitoxine-units. From to per cent of the original units with the euglobuline.

are recovered, while 10-20% are separatedoif guantitatively. The residue is res;

90 barium sulphate is filtered oil or removed by A chemical test shows the total absence of all inorganic and organic accompanying substances of. the original serum, such as chlorides, phosphates, carbonates, sulphates, phosphatids, cholesterin, carbon-hydrates,

moses, coloring matter and of albuminc and euglobuline.,- Y

Emample with pneumococci scm-mtlhis serum is treated inthe same manner, the final product showing the following characterisammo'nia, urea, uric acid, creatine, creatinine, tics and the like, of amino-acids, peptones, albu- Total proteids 7.57% Albumin .1. 17% Globuline 6. 4%;4. 7% pseudoglobuline-l- 1. 7% euglobuline Pseudoglobuline solution; V 9. 69% Yield 85% Concentration factor 1.8%

The chemical test shows complete absence of' all other substances except psendoglobulinc.

The pseudoglobuline thus obtained is particularly distinguished by the following properties as compared with the original serum It constitutes a colorless liquid, even at high protein-coneentrations which does not undergo any further changes in regard to its colloidal distribution and accordingly in re gard to its valuation, inasmuch as the labile euglobuline with the antigen residues, the l'ipoids, and the like cannot exercise any. further action by impairingthe degree of dispersion, and by producing the otherwise resultin g weakening of the sera. The viscosity of the pure globuline has been reatly reduced by the same reasons, and the a sorbing qualities are correspondingly increased. This very thorough purification of the'pseudoglobuline can only be effected by the electroosmotic method (electro-dyalitic fractionization), by means of which only lyophilous albumine+pseudoglobulines or pseudog'lobuline and lyophobous euglobuline and antigenresidues will remain. The perfect separation g of the two final fractions is characterized by amaximum of albuminous flocculation at the iso-electri'c point of the euglobulin (6.4 P

The pseudoglobnlines obtained in this manner containing the totality of the desired protective or curative bodies in a pure condition exhibit the same curative and protective effects as the original serum, but they are free 'from the drawbacks thereof, and aretherefore greatly superior to such sera in various respects. The specific pseudoglobulme-solu- 'tions thus obtained do not cause any serum disease and do not produce anaphylaxia even upo'n repeated administration. They are superior by their permanent keeping qualities, and they do not lose their curative efi'ects.

' In view ofall these facts their action is much quicker even in subcutaneous injection; nor do these injections produce pain or local irritative reactions at the point of injection. Particularly in the case ofthe. manufacture 'of highly concentrated immune sera the-relatively low contents of albuminous matter and the. low degree of viscosity constitute a very important progress in the particular art. The I speci fie pseiuloglolmlines obtained in the numncr descrlbed by the elcctro-osmohc method represent the efiic-ient principle, that is to say,

nous, and other inactive constituents of the. I

serum.

The invention is,-of course, not to be restricted to the particular features and steps, herein described'and specified by way of exemplification, but it is susceptible of modifications, except as will otherwise appear from the appended claims. 1

We claim 1, 'The method of purifying animal protective and curative'sera. which consists in treating such sera with a salt, thereby salting out and precipitating the globulines, dissolving the precipitate in water,flrcmoving the remaining precipitant therefrom, then sulnnitting the remaining globulivie-solution to electro-osmosis, and continuing the treatment, until at the isoelectric point the eulobuline has become deposited, and separatmg said deposit from the pseudo-globuline.

'2. The method of purifying animal protective and curative sera, which consists in treating such sera with an excess: of a sulphate, thereby salting out and precipitating the globulines, treating said precipitate with water and with barium-acetate, thereby precipitating bariumsulphate,' separating the barium sulphate from the globuline-solution, and submitting said solution to electro-osmosis, and continuing the treatment, until at the isoelectric point of 6.4 P concentration ofalbumin, and substantially incapable of flocculation 'at the iso-electric point of the globulines.

LUDWIG FEJES. DB. BRUNO BUTT-CHER 

